EP1076714A1 - Elaboration de cellules productrices retrovirales a partir de vecteurs adenoviraux et retroviraux - Google Patents

Elaboration de cellules productrices retrovirales a partir de vecteurs adenoviraux et retroviraux

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Publication number
EP1076714A1
EP1076714A1 EP99920141A EP99920141A EP1076714A1 EP 1076714 A1 EP1076714 A1 EP 1076714A1 EP 99920141 A EP99920141 A EP 99920141A EP 99920141 A EP99920141 A EP 99920141A EP 1076714 A1 EP1076714 A1 EP 1076714A1
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Prior art keywords
cells
vector
mlv
retroviral
gene
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German (de)
English (en)
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Xinli Lin
Jordan J. N. Tang
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Oklahoma Medical Research Foundation
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Oklahoma Medical Research Foundation
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10344—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13051—Methods of production or purification of viral material
    • C12N2740/13052—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • MMV Moloney murine leukemia virus
  • MLV-based vectors offer highly efficient chromosome integration, thus, the therapeutic genes are transmitted to the progeny cells
  • MLV-based gene delivery methods are largely limited to ex vivo protocols
  • a combination of adenoviral and retroviral vectors used to construct second generation packaging cells that deliver marker genes to target cells is described.
  • a vector based upon Moloney murine leukemia virus (MLV) was used to deliver marker genes, and an adenovirus-based delivery system was used to deliver MLV structural genes (gagpol and env) to cultured cells.
  • the procedure transformed the cells into new retroviral producer cells, which generate replication-incompetent retroviral particles in the culture supernatant for transferring marker genes to target cells.
  • the titer of the retroviral- containing supernatant generated from the second generation producer cells reached above 10 5 cfu/rnl, which is comparable to the MLV-based producer cell lines currently used in human gene therapy trials.
  • Additional adenoviral/MLV-based vectors were constructed with increased safety, by replacing the 5'-LTR down to the primer binding site in pPAM3 and replacing it with a CMV promoter.
  • Adenoviral vectors for delivering HIV-1 gagpol genes and marker genes in a lentivurs construct were made.
  • the examples demonstrate the construction of these vectors as well as delivery and expression of the thymidine kinase gene and killing of tumors in mice following gancyclovir administration.
  • the vectors and procedures are adaptable for human gene therapy in which the new producer cells are transplanted into patients for continuous gene transfer.
  • Figures 1A-1E are schematics of recombinant adenovirus gene constructs. From top to bottom, the constructs are: a) pPAM3, the parental MLV vector; b) p ⁇ El A-N/B, which contains the gagpol 'env genes and a polyadenylation signal; c) Ad-N/H-1 which contains the gagpol-env genes and not a polyadenylation signal; d) Ad-gp2, which contains the MLV gag- pol genes; and e) Ad-env3, which contains the MLV env gene, mu is a map unit of adenovirus Ad-5.
  • Figure 2 are schematics of the construction of vector pCA3-PAM3, showing elimination of the LTR and PBS site while retaining the SD site in vector pCA3-PAM3.
  • the adenovirus vector pCA3 is drawn in the reverse direction. 1 u 9.8 u in the figure represent the map unit of the adenovirus.
  • Figures 3 A, 3B and 3C are schematics of the construction of adenovirus vectors that deliver HIV-gagpol and marker genes.
  • Figure 3 A pCA14- ⁇ R8.2, which is an adenovirus vector that delivers HIV-gagpol gene under the control of a CMV promoter, is derived from vector pCMV- ⁇ R8.2.
  • adenovirus vector p ⁇ El A-v653-RSN which contains HIV-based vector sequences, delivers NEO-marker gene, is derived from v653-RSN.
  • Figure 3C p ⁇ El A-v653-GFP is the same as B, except that a CMV-GFP construct has been used to replace the SL3-NEO. 1 u, 9.8 u, map unit of adenovirus Ad-5.
  • Figures 4 A and 4B are schematics of constructs of retroviral vectors for delivering the TK and vhs genes.
  • Figure 3 A shows the construction of vector Lxpsp-HyTK. The unique restriction sites, Xhol and BamHI, are shown.
  • Figure 3B shows the dual expression vector pL-HyTK-mGM-CSF.
  • human primary cells To be converted to producer cells, human primary cells must acquire the therapeutic or marker gene and MLV structural genes gag, pol, and env.
  • the former are delivered by a conventional MLV-based vector while the latter are delivered by adenoviral vectors.
  • Different existing gene delivery systems are combined to produce new, and possibly more powerful, gene delivery constructs.
  • Adenoviral vectors to transfer MLV packaging genes to establish cell lines and primary cells.
  • Adenoviral vectors are an attractive choice for the transfer of MLV gag-pol-env genes into target cells since they have proven gene transfer efficiency and can accommodate a large insert.
  • the transferred structural genes are expressed by the new producer cells, and the MLV particles are apparently assembled with packaged marker genes, which are capable of transducing new target cells.
  • the adenoviral vectors transduce naive cells into transient packaging cells.
  • the titer of the new packaging cells reaches above 10 5 cfu/ml, which is comparable to MLV packaging cell lines currently used in the ex vivo procedures.
  • adenoviral vectors carrying MLV structural genes were able to rescue two different MLV-markers (pLN and pLNPOZ) in three different cells lines (NTH 3T3, Hela, and H9).
  • the pools of retroviruses transduced cells were also tested for adenoviral infection. This method is expected to produce lower titers but it is nevertheless a closer simulation for certain clinical applications, in which primary cells are first transduced with retroviral vectors, selected, then infected with adenoviral vectors. The results show that the vectors work reasonably well even with this rapid procedure. Moreover, co-infection by both retroviral and adenoviral vectors without selection also works.
  • the new producer cells transduced by adenoviral and retroviral vectors from primary cells may be introduced into patients to deliver therapeutic genes to target cells in vivo
  • Such a protocol has the advantage of a continuing in vivo gene transfer over the lifetime of the 'producer cell' This is also a safe procedure because both MLV and adenovirus-based gene delivery has been widely used in human gene therapy clinical trials (Blaese RM, et al. Science 1995, 270 470-474 Blaese RM, et al Hum Gene Ther
  • adenovirus vectors ( Figure 1) were constructed from adenoviral vector p ⁇ ElAsplA (p ⁇ ElA is used as prefix for the derived vectors in Fig. 1).
  • Vector p ⁇ ElA-N/B ( Figure lb) was obtained by inserting an 8.3 kb NheVBstX X07 I fragment from pPAM3, (Miller AD, et al. Mol Cell Biol 1986; 6: 2895-2902.) which contains the MLV left-side LTR, gagpol/env
  • Vector p ⁇ ElA-N/H (Ad-N/H-1 in Figure lc) was constructed by inserting a 7.9-kb Nhe ⁇ /Hpal fragment from pPAM3, which contains MLV 0 LTR, and gagpol/env genes without polyA signal, into the EcoRV site of p ⁇ lAsplA.
  • Vector p ⁇ l A-gp (Ad-gp2 in Figure 3d) contains MLV gag-pol genes derived from p ⁇ l A-N/B in which an EcoRI fragment at the 3 '-end containing part of the MLV env was deleted.
  • 5 Vector p ⁇ l A-env (Ad-env in Figure le) was produced as follows: an_YZ> ⁇ I site in the left LTR of pPAM3 was eliminated by partial XbaX digestion and Klenow fill-in. The resulting plasmid was digested with Pvul/Xba ⁇ , filled-in with Klenow, and the vector was religated. These steps eliminated most of the gag-pol genes. The resulting vector was then cut with
  • 293 - ⁇ cells were transfected with pPAM3 and p ⁇ ElA-N/H by calcium phosphate transfection method (Graham, FL, van der Eb AJ. Virology 1973; 52: 456-467.) using a commercial kit from Promega (Perfection Mammalian Transfection System, Promega Co., Madison, WI,
  • MLV-based vectors were either transfected into an intermediate ecotropic packaging cell line GP+E-86 (Markowitz D, et al. J Virol 1988; 62: 1120-1124. Markowitz D, et al. Ann NYAcad Sci 1990; 612: 407-414.) using either calcium phosphate transfection as described above or lipofectamine transfection (Life Technologies, Inc., GibcoBRL, Gaithersburg,
  • each of the 4 vectors was co- transfected with either pBHGl 1 or pBHGlO (Bett AJ, et al. Proc NatlAcad Sci USA 1994; 91: 8801-8806.) into 293 cells by calcium phosphate method basically as described.
  • pBHGl 1 or pBHGlO Bett AJ, et al. Proc NatlAcad Sci USA 1994; 91: 8801-8806.
  • Hela or 3T3 cells were grown in DMEM/10% FCS and then plated in 6-well plates with 2.4 x 10 5 cells/well at day one to reach about 40-60% confluence at day two. All culture media containing the MLV virus-like particles were centrifuged at 1,500 rpm for 10 min in a Beckman GPR centrifuge. To avoid possible contamination from producer cells, only the top layers of the centrifuged supernatant were used for infection. Frozen supernatants were thawed and centrifuged only, while fresh supernatants were centrifuged and then filtered through 0.45 ⁇ m filters before being used for infection.
  • the cells were separately infected with the supernatants from MLV producer cells PA317-LN or PA317-LNPOZ in the presence of 8 ⁇ g ml polybrene and adenoviral vectors Ad-N/H-1, Ad-gp2, and Ad-env3. Cells were washed with medium three times after infection to completely remove residual retroviral particles and then cultured for two additional days.
  • 3T3 cells were plated in 6-well plates as described above the day before infection. After infection with different dilutions of supernatants in the presence of polybrene, the cells were incubated in a CO 2 - incubator for 24 h before being transferred into 10-cm dishes and selected with G418 (0.75 mg/ml active G418). Selection media were changed after each period of 2 to 3 days, and colonies were stained with either crystal violet (0.1%) in 20%) ethanol) or X-gal before counting. Marker rescue assays
  • Controls include 3T3 and Hela cells infected with supernatants from either P A317-LN or PA317-LNPOZ alone, or infected with adenoviral vectors alone. Additional controls include infection of 3T3-LN, 3T3-LNPOZ and Hela-LN clones with either Ad-gp2 or Ad-env3 alone. All of these controls produced no count when titered on 3T3 cells.
  • NTH 3T3-pLN cells Genomic analysis of NIH 3T3-pLN cells Three different cells were used to analyze the genomic integration patterns of retroviral vectors. NTH 3T3 cells were used as negative controls.
  • Genomic DNA was purified from cells (3 x 10 6 ) using a commercial kit (Wizard Genomic DNA Purification System, Promega) according to manufacturer's instructions. The
  • DNA (7 ⁇ g) from these three cell lines was digested with Bam HI, which is not cut in pLN gene, and then separated on 1% agarose gels. The gels were cut into 9 equal sections from 3 to 20 kb, and about 5 ⁇ l from each section was used as templates for PCR amplifications.
  • Two internal primers of pLN were used for the PCR: forward primer
  • PCR was performed with a "touch-down” method, with separation for 40 seconds at 94°C, extension for 60 seconds at 72°C, and various annealing temperatures of 55°C, 53°C, 50°C for 5 cycles each followed by 48°C, 46°C,
  • PBMCs Peripheral blood mononuclear cells
  • PHA-P phytohemagglutinin
  • continuous culture of the cells was done in the same media with interleukin-2 (human recombinant LL-2 from GibcoBRL, 200 u/ml).
  • PBMCs (9 x 10 6 in 5 ml) were infected with 5 ml of supernatant from PA317-LNPOZ (5.8 x 10 6 cfu/ml) in the presence of 8 ⁇ g/ml polybrene for 4 h. Cells were washed three times with PBS, and then resuspended in 6 ml of growth medium with LL-2. Cells were seeded into a 24-well plate with 1 ml aliquots, and infected with Ad-N/H-1 with MOI of 0, 5, 10, 20, 50, and 100.
  • Example 1 Construction and Functional tests of adenoviral vectors.
  • the four different fragments of MLV genes which were cloned separately into adenoviral vector p ⁇ ElAsplA (p ⁇ El A is used as prefix for the derived vectors in Figure 1) were tested for their functionality
  • Vector p ⁇ ElA-N H which contains all 3 MLV structural genes, was transfected by calcium phosphate coprecipitation method into 293 ⁇ cells, which contain an integrated marker ⁇ -galactosidase gene originally transduced from a retroviral vector. The expression of MLV structural genes would rescue the marker gene, and package it into the resulting retroviral vectors.
  • PpaM3 180 ⁇ 58 * Titers are expressed as colony forming unit (cfu) per ml and each set of data represents 5 independent experiments.
  • Ad-N/H- 1 has the capacity to deliver all MLV gag-pol-env genes.
  • recombinants between pBHGlO and either p ⁇ ElA-gp or p ⁇ El A-env were also obtained.
  • These recombinant vectors separately deliver MLV gag-pol- env genes (Ad-gp2) and env gene (Ad-env3).
  • Example 2 Infection of NIH3T3 cells with recombinant adenoviral vectors results in the expression of MLV structural genes.
  • MLV structural gene products in 3T3 cells was monitored by Western Blot, using an anti-MLV antiserum, after infection with CsCl purified recombinant adenoviruses Ad-N H-1, Ad-gp2, and Ad- env3.
  • Three known MLV protein bands (Felsenstein KM, et al. J Virol 1988; 62: 2179-2182; Miller AD, et al. J Virol 1984; 49: 214-222) gp80 (env), p65 (gag) and p30 (gag), whose positions in Western blot are marked by a retroviral packaging cell line P A317 (Miller AD, et al. Mol Cell Biol
  • Ad-gp2 Cells receiving only gag-pol genes (Ad-gp2) produced only the expected proteins p65 and p30. Likewise, infection of Ad-env3 produced only gp80. Coinfection of Ad-gp2 and Ad-env3 produced all 3 bands. Reverse transcriptase (RT) assays were performed to assess the virus production in the supernatants. The results showed that while the supernatants from PA317 clones contains 0.25 ng/ml RT, the supernatant from Ad-N/H-1 and Ad-gp2 infected cells contain RT from 0.02 to 8 ng/ml, depending on the dose and duration of the adenovirus infection.
  • RT Reverse transcriptase
  • Example 3 Cotransduction of cells with retroviral and adenoviral vectors to generate new producer cells.
  • the marker genes neoR (from pLN) and neoR + lac z (from pLNPOZ) were first transduced into NTH 3T3 and Hela cells, and high titer clones were selected.
  • C 3T3 cells in 6-well plates were infected with CsCl purified Ad-N/H-1 with MOI of 0, 7 and 14 respectively The resulting supernatants were then titered on NIH 3 T3 cells.
  • d H9-LN were infected with Ad-N/H-1 with MOI of 0, 140, and 280, respectively The resulting supernatants were then titered on NTH 3T3 cells.
  • Table 2 also shows that the titers are zero without recombinant retrovirus.
  • NTH3T3-LN, NTH3T3-LNPOZ and Hela-LN clones were infected with either Ad-N/H-1 or Ad-gp2 plus Ad-env-3. Supernatants from the resulting infections were then used to infect NTH3T3 cells and selected with G418. The neomycin-resistant colonies were then counted and expressed as cfu per ml.
  • Table 3 shows that the titers for the 3T3 clones ranged from 3 x 10 4 to 3 x 10 5 cfu/ml and for HeLa clones ranged from 8 x 10 3 to 1 x 10 4 cfu/ml.
  • the sensitivity of HeLa cells to the cytopathic effect of Ad-N/H-1 may account for the titer being lower than that from Ad-N/H-1, possibly because of the uneven transfer and expression of gp2 and env3 genes in individual producer cells.
  • PBMCs peripheral blood mononuclear cells
  • Retroviral vector-containing cells were cultured in 6-well plates and infected with Ad-N/H-1 as described in Methods. Supernatants were collected 2 days after the infection (Sup-I), and cells were trypsinized and transferred to 10-cm plates and culture continued without selection, the resulting supernatants were collected again after 2 days (Sup-II), and cells were split and cultured again. The final supernatants were collected after another 2 days (Sup-III).
  • the 5'- primer (CAM3P1) is: 5'-GTTAAC CAG GGA CCA CCG ACC CAC CAC CGG GAG GTA AGC TGG GtT GCA GCA TCG-3 1 (SEQ ID NO. 3), where the italic letters represent an installed Hpal site and the small letter t is a mutation changed from C that eliminated the 5' side Pstl site.
  • the 3 '-primer (CAM3P2) is: 5'-CTGCAG AGC AGA AGG TAA CCC-3' (SEQ TD NO. 4), where the italic letters are the original Pstl site shown in Figure 2.
  • the gene delivery vectors are HIV based instead of MLV based.
  • the construction of three vectors are described below and with reference to Figures 3 A, 3B and 3C.
  • Figures 3 A, 3B and 3C show the structure of three adenovirus vectors for delivering HIV-1 gagpol genes and marker genes in the lentivirus construct.
  • a vector pCMV ⁇ R8.2 from Dr. Inder Verma of The Salk Institute for Biological Studies.
  • the resulting vector was cut with Notl, and a 7.4 kb fragment, which contains gagpol, rev, Vi Vpr, Vpu, RRE, and Nef genes, was Klenow filled-in, and cloned into the EcoRV site of pCA14 (Microbix Biosystems Inc., Toronto, Canada).
  • the resulting vector, pCA14 ⁇ R8.2 ( Figure 3A), is similar to the original pCMV ⁇ R8.2 in that the HTV structural and accessory genes are under the control of a CMV promotor.
  • Vector pCA14 ⁇ R8.2 was co-transfected with pBHGlO into 293 cells using calcium phosphate method as described (1), and the recombinant virus, Ad- CA14 ⁇ R8.2, has been obtained and is in the process of plaque-purification and characterization.
  • Vector p ⁇ ElA-V653-RS ⁇ (Fig. 2B), an adenovirus vector, was designed to deliver a marker (NEO) gene in the HIV genomic construct.
  • the parent plasmid v653-RSN was obtained from Dr. Joseph Sodroski. Plasmid v653-RSN was cut with ClaX, then partially cut with NdeX. The resulting 7.6- kb fragment was recovered and filled-in with Klenow before it was cloned into the EcoRV site of p ⁇ 1 sp 1 A (Microbix) to form the plasmid p ⁇ 1 A- v653-RSN. The insert size (7.6 kb) was well below the limit of 7.9 kb for recombination with pBHGlO.
  • Vector p ⁇ lA-v653-RSN was co-transfected into 293 cells with pBHGlO and the recombinant virus, Ad-v653-RSN, obtained and plaque purified and characterized.
  • Vector p ⁇ ElA-V653-GFP Figure 3C
  • GFP green fluorescent protein
  • An MLV-based vector, pLXSN was digested with EcoR I, and then treated with Klenow following ligation to eliminate the EcoR I site.
  • the resulting vector was cut with Bam HL/Nco I, filled-in with Klenow, and religated to eliminate most of the Neo sequence in the original vector.
  • the new vector called pLX was cut with Xho I Bam HI.
  • An Xho I Bgl II polylinker fragment from psp73 (Promega) was then cloned into the Xho
  • Lxpsp contains the MLV-LTR plus a polylinker site for cloning.
  • a 2.9 kb Xho I/Bgl II fragment from vector tgCMV/HyTK was then cloned into the EcoR I site of Lxpsp, resulting in Lxpsp-HyTK ( Figure 4A), which contains a hygromycin B- thymidine kinase fusion gene under the control of a CMV promotor in an
  • Lxpsp-HyTK was transfected into an ecotropic packaging cell line GP+E-86 by calcium phosphate transfection, and the supernatant used to infect an amphotropic packaging cell line PA317. High-titer clones of PA317-HyTK were then obtained by colony selection and titration.
  • a dual- expression vector pLmGM-CSF-HyTK ( Figure 4B) was cloned by inserting a
  • mGM-CSF granulocyte-monocyte colony stimulating factor
  • the mGM-CSF gene is obtained from vector pNGVLl -mGM-CSF, which was obtained from the National Gene Vector Laboratory (NGVL) plasmid repository at the University of Michigan. This vector is an "enhanced" version of the HyTK vector, due to the immune-stimulating effect of GM-CSF,
  • PA317-HyTK cells were cloned, and the high-titer clones selected.
  • the results showed that the parental cell lines, NIH-TK " , K-Balb, and 80TTB, are all resistant to GCV and can only be killed at 1 mM of GCV concentration.
  • the high titer t#-containing clones of these cells which include HyTK and HyTK-mGM-CSF, are sensitive to GCV and can be killed at 10 DM of GCV concentration.
  • Balb/c mice were used as animal models and the tumor cell line K- Balb was used as tumor inoculation cells.
  • each mouse was injected with tumor cells only (2x10 6 tumor cells/inoculation) in the left flank and tumor (2x10 6 cells) plus packaging cells (8xl0 5 cells) in the right flank. Tumors were grown to almost full size at day twelve, and ganciclovir (150 mg/ml, twice daily for five days) was injected intraperitoneally.

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Abstract

L'invention concerne une combinaison de vecteurs adénoviraux et rétroviraux utilisée pour élaborer des cellules d'encapsidation de la seconde génération qui transfèrent des gènes marqueurs à des cellules cibles. On a utilisé un vecteur reposant sur le virus de la leucémie murine pour transférer les gènes marqueurs, d'une part, et un système de transfert à base adénovirale pour transférer les gènes structuraux de ladite leucémie (gagpol et env) à des cellules mises en culture, d'autre part. Via cette procédure, on a pu transformer des cellules en nouvelles cellules productrices rétrovirales, générant des particules rétrovirales à incompétence de réplication dans le surnageant de culture, pour assurer le transfert de gènes marqueurs à des cellules cibles. Le titre du surnageant rétroviral issu des cellules productrices de la seconde génération a ainsi dépassé 105 cfu/ml, ce qui est comparable au niveau des lignées cellulaires productrices reposant sur le virus de la leucémie murine, actuellement utilisées dans les essais de thérapie génique chez l'homme. On peut utiliser ce type de vecteur et de procédure pour la thérapie génique expérimentale chez l'homme, ce qui revient à transplanter aux patients les nouvelles cellules productrices pour les besoins d'un transfert génique continu.
EP99920141A 1998-04-29 1999-04-29 Elaboration de cellules productrices retrovirales a partir de vecteurs adenoviraux et retroviraux Withdrawn EP1076714A1 (fr)

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WO2000018240A1 (fr) * 1998-10-01 2000-04-06 University Of Southern California Systeme de transport de gene et procedes d'utilisation associes
US7052904B2 (en) * 2000-01-31 2006-05-30 The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services Hybrid adeno-retroviral vector for the transfection of cells
WO2002061104A2 (fr) * 2001-01-30 2002-08-08 The Government Of The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Vecteur adenoviral hybride
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CA2327444A1 (fr) 1999-11-04
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AU3770999A (en) 1999-11-16
WO1999055894A1 (fr) 1999-11-04
AU758155B2 (en) 2003-03-13
JP2002512805A (ja) 2002-05-08

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